Collision cooling of ions is now widely used for the purpose of improving the quality of the ion beams. Cooling can be accomplished in an RF only ion guide as disclosed in U.S. Pat. No. 4,963,736 to Douglas, et al. or in gas chamber, that do not include RF rods. Both these techniques provide a buffer gas, and the presence of the buffer gas slows down the ions and, in the case of the RF-ion guide, can lead to reduction of the size of the ion beam. The process may also cool down internal vibration and other degrees of freedom of the ions.
In some cases the ions acquire a high degree of internal excitation during ionization or other processes. If left excited, the ions will eventually fragment; this process is called metastable fragmentation. Metastable fragmentation is one of the main reasons for poor quality spectra of large proteins and DNAs using MALDI (See, for example, A. V. Loboda, A. N. Krutchinsky, M. Bromirski, W. Ens, K. G. Standing, “A tandem quadrupole/time-of-flight mass spectrometer (QqTOF) with a MALDI source: design and performance”, Rapid Commun. Mass Spectrom. 14, 1047 (2000))]. Some other ionization methods (surface ionization mass spectrometry SIMS, fast atom bombardment FAB, Laser ablation LA, electron impact EI, etc) have similar problems and the present invention is generally applicable to such other methods. However, the present invention is primarily intended for application to MALDI sources and the invention will be described primarily in relation to MALDI sources. Metastable fragmentation means that ions can spontaneously fragment at any time and at any location in a mass spectrometer instrument, and hence can give poor spectra.
Because of this limitation, two types of axial MALDI TOF (Time of Flight) systems now exist on the market: linear MALDI TOF and reflectron MALDI TOF. In a linear MALDI TOF, ions are pulsed from an extraction region into a linear flight tube, and the ions are detected at the end of the flight tube. The time of flight through the flight tube depends upon the initial energy given to the ions in the extraction region and the ions' mass to charge ratio. As ions have some energy and velocity before the extraction pulse is applied, this motion is reflected in the velocity of ions m/z ratio as they travel through the flight tube. The overall effect is to degrade the resolution and accuracy of a linear time of flight instrument. For this reason, reflectron MALDI TOF instruments were developed. In a reflectron MALDI TOF, ions are again pulsed out of an extraction region and are provided with a pulse of energy. However, after traveling through the first part of the flight tube, the ions enter a reflection region where a field is applied to reflect the ions back to a location beside the original extraction region. The overall effect, approximately, is to negate or at least reduce the effect of any original ion motion in the direction of ion travel, so that reflectron TOF instruments have excellent resolution and mass accuracy.
Because of the different characteristics of linear and reflectron TOF instruments, metastable fragmentation has quite different effects in these two instruments. In a linear MALDI TOF instrument, although it has limited resolution and mass accuracy, it is much more tolerant of metastable fragmentation. This is because once the ions leave the short extraction region, they enter a field free drift chamber. If a metastable ion fragments in the drift tube the velocities of the fragments do not change significantly from the velocity of the original ion. Hence, the fragments will still arrive at the detector at the same time as the unfragmented ions, and there is little effect or degradation on the spectrum obtained.
In contrast, in a reflectron instrument, if metastable fragmentation occurs before or in the reflector, this will cause the fragment to spend a different time in the drift chamber before reaching the detector, causing significant degradation of the spectrum. It is for this reason that linear MALDI TOF is used where metastable fragmentation is perceived to be a potential problem.
As a first approximation, a linear MALDI TOF device can tolerate metastable fragmentation that occurs after a few microseconds (the time it takes for ions to leave the extraction region), while a reflectron MALDI device can only tolerate the metastable fragmentation that has a time scale of approximately 100 microseconds (the time when the ions leave the reflector); The time scale of metastable fragmentation usually depends on the level of internal excitation of the ions, the higher the degree of excitation the faster the ion will fragment.
Collisional cooling of MALDI ions as disclosed in published International Patent Application No. WO99/38185 can cure the problem of metastable fragmentation to some extent. In one preferred embodiment the ions are cooled down at a pressure ˜10 mTorr. At this pressure the cooling time is about 100 μs. Thus, the fragmentation pattern in the spectra resembles the ones in Reflectron MALDI TOF, as some metastable fragmentation still occurs. The only difference is that the resolution and mass accuracy of the observed fragments in MALDI with collisional cooling stays the same as for the stable ions. Both fragments and primary ions leave the cooling stage cooled down and focused, prior to entry into the TOF section. As the ions are then cooled, no subsequent metastable fragmentation occurs in the TOF section.
As the cooling time is inversely proportional to the pressure another arrangement was disclosed in published International Patent Application No. WO99/38185. That arrangement has a cooling stage at a pressure of ˜1 Torr. The cooling time in this case is ˜1 As and this is short enough that fragmentation is substantially reduced. The spectra observed resemble the spectra from a linear MALDI TOF.
Unfortunately such a high pressure has the disadvantage that it can affect the ionization process resulting in cluster formation. Clusters of ions of interest with several matrix molecules begin to appear as the pressure increase. Since a typical MALDI sample has substances of interest embedded in the excess of the matrix molecules it has been speculated that the clusters represent the material that was cooled down too rapidly without allowing matrix molecules to “evaporate” from the analyte ions.